A practical molecular identification of nonfermenting Gram-negative bacteria from cystic fibrosis

Capizzani, Carolina Paulino da Costa; Caçador, Natália Candido; Marques, Elizabeth Andrade; Levy, Carlos Emílio; Tonani, Ludmilla; Torres, Lidia Alice Gomes Monteiro Marin; Darini, Ana Lúcia da Costa

doi:10.1016/j.bjm.2017.07.002

person Carolina Paulino da Costa Capizzani

schoolUniversidade de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Ribeirão Preto, SP, Brazil

person Natália Candido Caçador

schoolUniversidade de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Ribeirão Preto, SP, Brazil

person Elizabeth Andrade Marques

schoolUniversidade Estadual do Rio de Janeiro, Faculdade de Ciências Médicas, Departamento de Microbiologia, Imunologia e Parasitologia, Rio de Janeiro, RJ, Brazil

person Carlos Emílio Levy

schoolUniversidade Estadual de Campinas, Faculdade de Ciências Médicas, Departamento de Patologia Clínica, Campinas, SP, Brazil

person Ludmilla Tonani

schoolUniversidade de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Ribeirão Preto, SP, Brazil

person Lidia Alice Gomes Monteiro Marin Torres

schoolUniversidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Puericultura e Pediatria, Serviço de Referência de Fibrose Cística do Hospital das Clínicas, Ribeirão Preto, SP, Brazil

person Ana Lúcia da Costa Darini *

schoolUniversidade de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Ribeirão Preto, SP, Brazil

person*Corresponding author at: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Avenida do Café, s/n
o, Ribeirão Preto, SP, Brazil. E-mail:
aldarini@usp.br (A.L. Darini).

Conflicts of interest

The authors declare that they have no conflict of interest.

Primer Target	Primers	Annealing temperature (ºC)	Product size (bp)a	References
B. cepacia (Genomovar I)	BCRG11 BCRG12	62	492	Mahenthiralingam et al.²²
B. multivorans (Genomovar II)	BCRBM1 BCRBM2	62	714	Mahenthiralingam et al.²²
B. cenocepacia (Genomovar III-A)	BCRG3A1 BCRG3A2	62	378	Mahenthiralingam et al.²²
B. cenocepacia (Genomovar III-B)	BCRG3B1 BCRG3B2	60	781	Mahenthiralingam et al.²²
B. stabilis (Genomovar IV)	BCRG41 BCRG42	64	647	Mahenthiralingam et al.²²
B. vietnamiensis (Genomovar V)	BCRBV1 BCRBV2	62	378	Mahenthiralingam et al.²²
B. dolosa (Genomovar VI)	G6N BCR1	67	135	Vermis et al.²³
B. ambifaria (genomovar VII)	BCRGC1 BCRGC2	62	810	Coenye et al.²⁴
B. anthina (genomovar VIII)	BCRG81 BCRG82	61	473	Vandamme et al.²⁵
Complexo B. cepacia - Gene recA	BCR1 BCR2	58	1043	Mahenthiralingam et al.²²
A. xylosoxidans	AX-F1 AX-B1	56	163	Liu et al.²⁶
S. maltophilia	SM1 SM4	58	531	Whitby et al.²⁷
P. sputorum	spuF spur	63	813	Coenye et al.²⁸
Genus Ralstonia	RalGS-F RalGS-R	58	546	Coenye et al.²⁹
Genus Cupriavidus	ral2f ral2r	58-61	187	Barrett and Parker³⁰

Primers	Genus/species	Targets	Proposed modificationsa
	Literature	The present study
BCR1/BCR2	Bcc bacteria	Bcc bacteria	AT 55 ºC and 56 ºC
AX-F1/AX-B1	A. xylodoxidans	Achromobacter sp.	NMT
SM-1/SM4	S. maltophilia	S. maltophilia	NMT
spuF/spuR	P. sputorum	Pandoraea sp. + Ralstonia sp.	NMT
RalGS-F/RalGS-R	Ralstonia sp.	Ralstonia sp. + Cupriavidus sp.	NMT
ral2f/ral2r	Cupriavidus sp.	Cupriavidus sp. + Ralstonia sp. + Pandoraea sp. + Achromobacter sp.	AT 62 ºC

Bacteria tested	Genus/species Primers
	Bcc BCR1/BCR2	P. sputorum spuF/spuR	Ralstonia sp. RalGS-F/RalGS-R	A. xylosoxidans AX-F1/AX-B1	S. maltophilia SM-1/SM4	Cupriavidus sp. ral2f/ral2r
Reference strains
A. denitrificans LMG 1231	Negative	Negative	Negative	Positive	Negative	Positive a
A. piechaudii LMG 1873	Negative	Negative	Negative	Positive	Negative	Positive a
A. xylosoxidans LMG 1863	Negative	Negative	Negative	Positive	Negative	Positive a
A. dolens LMG	Negative	Negative	Negative	Positive	Negative	Positive a
A. insuavis	Negative	Negative	Negative	Positive	Negative	Positive a
A. mucicolens	Negative	Negative	Negative	Positive	Negative	Positive a
A. ruhlandii	Negative	Negative	Negative	Positive	Negative	Positive a
C. gillardi LMG 5886	Negative	Negative	Positive a	Negative	Negative	Positive
P. norimbergensis LMG 18379	Negative	Positive	Negative	Negative	Negative	Positive a
P. pnomenusa LMG 18087	Negative	Positive	Negative	Negative	Negative	Positive a
P. pulmonicula LMG 18106	Negative	Positive	Negative	Negative	Negative	Positive a
P. apista LMG 16407	Negative	Positive	Negative	Negative	Negative	Positive a
R. picketti LMG 5942	Negative	Positive a	Positive	Negative	Negative	Positive a
S. maltophilia LMG 958	Negative	Negative	Negative	Negative	Positive	Negative
B. cepacia 1254	Positive	Negative	Negative	Negative	Negative	Negative
B. multivorans 788	Positive	Negative	Negative	Negative	Negative	Negative
B. cenocepacia LMG 21462	Positive	Negative	Negative	Negative	Negative	Negative
B. cenocepacia 818 Gen. IIIA	Positive	Negative	Negative	Negative	Negative	Negative
B. cenocepacia 17604 Gen. IIIA	Positive	Negative	Negative	Negative	Negative	Negative
B. cenocepacia 842 Gen. IIIB	Positive	Negative	Negative	Negative	Negative	Negative
B. cenocepacia 805 Gen. IIIB	Positive	Negative	Negative	Negative	Negative	Negative
B. stabilis 790	Positive	Negative	Negative	Negative	Negative	Negative
B. stabilis 825	Positive	Negative	Negative	Negative	Negative	Negative
B. vietnamiensis LMG 10929	Positive	Negative	Negative	Negative	Negative	Negative
B. vietnamiensis 1109	Positive	Negative	Negative	Negative	Negative	Negative
B. dolosa LMG-18943	Positive	Negative	Negative	Negative	Negative	Negative
B. ambifaria LMG 19182	Positive	Negative	Negative	Negative	Negative	Negative
B. ambifaria ATCC-53266	Positive	Negative	Negative	Negative	Negative	Negative
B. ambifaria AMMD	Positive	Negative	Negative	Negative	Negative	Negative
B. anthina LMG-20980	Positive	Negative	Negative	Negative	Negative	Negative
B. anthina LMG-16670	Positive	Negative	Negative	Negative	Negative	Negative
B. pyrrocinia ATCC-39227	Positive	Negative	Negative	Negative	Negative	Negative
B. pyrrocinia LMG-14191	Positive	Negative	Negative	Negative	Negative	Negative
Isolates
Bcc (n = 91)	Positive	Negative	Negative	Negative	Negative	Negative
Achromobacter sp. (n = 85)	Negative	Negative	Negative	Positive	Negative	Positive a
S. maltophilia (n = 12)	Negative	Negative	Negative	Negative	Positive	Negative
Ralstonia sp. (n = 10)	Negative	Positive a	Positive	Negative	Negative	Positive a
Pandoraea sp. (n = 2)	Negative	Positive	Negative	Negative	Negative	Positive a
Cupriavidus sp. (n = 1)	Negative	Negative	Positive a	Negative	Negative	Positive

[TFN1] ^a
Size of amplified product by PCR.